Tertiary Structure

The tertiary structure of a protein refers to the arrangement of amino acid side chains in the protein. Generally, the information for protein structure is contained within the amino acid sequence of the protein itself. This important principle of biochemistry was first determined by the biochemist Christian Anfinsen in studies of the enzyme ribonuclease. Ribonuclease catalyzes a simple hydrolysis of ribonucleic acid. The native enzyme has 124 amino acids; 8 of these are cysteines, forming 4 disulfide bonds. When ribonuclease was treated with mercaptoethanol to destroy the disulfide bonds and urea to disrupt its secondary and tertiary structure, all enzymatic activity was lost. Physical methods showed that this denatured form of ribonuclease had lost all detectable secondary and tertiary structure, although its amino acid sequence (primary structure) was intact. Anfinsen then slowly removed the urea and mercaptoethanol, then exposed the solution to air to reoxidize the cysteine pairs to disulfides. The renatured enzyme had full activity, leading to the conclusion that all the information required for the enzyme's three‐dimensional structure was present only in the linear sequence of amino acids it contained and that the active structure of the enzyme was the thermodynamically most stable one.

This principle has been validated many times. For example, several enzymes, including ribonuclease, have been synthesized chemically from amino acids. The synthetic enzymes are fully active. Another validation has come from biotechnology. For example, human insulin, a hormone rather than an enzyme, can be made by yeast carrying the appropriate genes. Insulin made this way is indistinguishable from natural human insulin and is used extensively in treating diabetes.

Protein tertiary structure. Protein tertiary structures are the result of weak interactions. When a protein folds, either as it is being made on ribosomes or refolded after it is purified, the first step involves the formation of hydrogen bonds within the structure to nucleate secondary structural (alpha and beta) regions. For example, amide hydrogen atoms can form H‐bonds with nearby carbonyl oxygens; an alpha helix or beta sheet can zip up, prompted by these small local structures.

Hydrophobic interactions among the amino acid side chains also determine tertiary structure. Most globular proteins have their hydrophobic side chains, for example, those of phenylalanine, valine, or tryptophan, located on the inside of the protein structure. Conversely, the hydrophilic amino acids, such as glutamic acid, serine, or asparagine, are generally found on the outside surface of the protein, where they are available for interaction with water. Alternatively, when these groups are found on the inside of soluble proteins, they often form charge‐charge interactions, or salt bridges, bringing a positively charged side chain (such as Arg) close to a negative one (such as Glu).

In membrane proteins, these general principles are reversed: The hydrophobic amino acid side chains are found on the outside of the protein, where they are available to interact with the acyl groups of the membrane phospholipids, while the hydrophilic amino acids are on the inside of the protein, available for interacting with each other and with water‐soluble species, such as inorganic ions. Membrane proteins are usually synthesized on membrane‐bound ribosomes to facilitate their proper assembly and localization. Some membrane‐bound proteins are synthesized on cytoplasmic ribosomes, with their hydrophobic residues inside the molecule, and they undergo a refolding when they later encounter the membrane where they will reside . Van der Waal's forces are important for a protein achieving its final shape. Although they are individually very weak, the sum of these interactions contributes substantial energy to the final three‐dimensional shape of the protein.

Proteins may assist the folding of other proteins. Although the native, active structure of a protein is thought to be the most stable one thermodynamically, it isn't always achieved in high yield when a protein is allowed to fold on its own. This can be true whether the protein is synthesized in vitro or in vivo—outside or inside a living body. Most cells contain a variety of proteins, called chaperonins, which facilitate the proper folding of newly synthesized or denatured proteins. Chaperonins use ATP energy to assist the refolding of proteins. Because proteins are often denatured by heat (think of a hard‐boiled egg), many chaperonins are expressed at high levels during heat shock of cells. Fever is one physiological heat shock, and chaperonins are among the proteins that protect cellular proteins from denaturing during a fever.

Usually, disulfide bonds form after a protein has achieved a final tertiary structure. Because they are so strong, the premature formation of an incorrect hydrogen bond could force a protein into an inactive tertiary structure. For example, if the disulfides of ribonuclease are allowed to form when the protein is in a denatured state, less than 1% of the enzyme activity is recovered, indicating that only a small minority of the disulfides are correct.

In contrast, when the protein is allowed to form the proper tertiary structure before disulfide formation, essentially all the enzymatic activity is recovered. The disulfide interchange enzyme acts on newly made proteins, catalyzing the breakage and rejoining of disulfides in a protein. Combined with the action of the chaperonins, the enzyme helps the protein achieve its final, native state, with all the disulfides formed appropriately.